1. Field of the Invention
This invention relates to a measuring method and a measuring system where a light beam is caused to be reflected in total internal reflection at an interface between a film layer in contact with an object to be measured such as a sample and a dielectric block to generate evanescent waves, and the change in the intensity of the light beam reflected in total internal reflection is measured to analyze the sample.
2. Description of the Related Art
As a measuring system using evanescent waves, there has been known a surface plasmon sensor. In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon. The surface plasmon sensor analyzes the property of the sample utilizing a phenomenon that such surface plasmon is excited by light waves. There have been proposed various types of surface plasmon sensors. Among those, one employing a system called “Kretschmann configuration” is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism. A metal film is formed on one face of the dielectric block and is brought into contact with a sample. A light source emits a light beam. An optical system causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface. A photodetector means detects the intensity of the light beam reflected in total internal reflection at the interface. A measuring means detects a state of surface plasmon resonance on the basis of the result of detection of the photodetector means.
In order to obtain various angles of incidence of the light beam to the interface, a relatively thin incident light beam may be caused to impinge upon the interface while deflecting the incident light beam so that the angle of incidence changes or a relatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light so that components of the incident light beam impinge upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle which varies as the incident light beam is deflected may be detected by a small photodetector which is moved in synchronization with deflection of the incident light beam or by an area sensor extending in the direction in which reflected light beam is moved as a result of deflection. In the latter case, the light beam which is reflected from the interface can be detected by an area sensor which extends in directions so that all the components of light reflected from the interface at various angles can be detected.
In such a plasmon resonance sensor, when a light beam impinges upon the interface at a particular angle of incidence θsp not smaller than the angle of total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample by the evanescent waves. When the wave number vector of the evanescent waves is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the light beam to impinge upon the interface in the form of p-polarized light.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                                          ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and εm and εs respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant εs of the sample is known, the refractive index of the sample and the like can be calculated on the basis of a predetermined calibration curve and the like. Accordingly a property related to the dielectric constant εs of the sample or the refractive index of the sample can be detected by detecting the angle of incidence θsp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops (this angel θsp will be referred to as “the attenuation angle θsp”, hereinbelow).
As a similar apparatus utilizing the evanescent waves, there has been known a leaky mode sensor described in, for instance, “Surface Refracto-Sensor using Evanescent Waves: Principles and Instrumentations” by Takayuki Okamoto, Spectral Research, Vol. 47, No. 1 (1998), pp. 19-28. The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one face of the dielectric block. An optical waveguide layer is formed on the clad layer and is brought into contact with a sample. A light source emits a light beam. An optical system causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface. A photodetector means detects the intensity of the light beam reflected in total internal reflection at the interface. A measuring means detects a state of excitation of the waveguide mode on the basis of the result of detection of the photodetector means.
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, only light having a particular wave number and impinging upon the optical waveguide layer at a particular angle of incidence comes to propagate through the optical waveguide layer in a waveguide mode after passing through the clad layer. When the waveguide mode is thus excited, almost all the incident light is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer in a waveguide mode depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the angle of incidence θsp at which the attenuation in total internal reflection occurs.
The surface plasmon sensor and the leaky mode sensor are sometimes used in random screening for finding an analyte combined with a predetermined ligand in the field of pharmacy or the like. In this case, the ligand is fixed on the film layer (the metal film in the case of the surface plasmon sensor, and the clad layer and the optical waveguide layer in the case of the leaky mode sensor), and buffers (sample liquid) containing therein various analytes are added to the ligand. Then the attenuation angle θsp is repeatedly measured each time a predetermined time lapses. When the analyte in the buffer is combined with the sensing material, the refractive index of the ligand changes with time due to combination with the analyte. Accordingly, by measuring the attenuation angle θsp, at which attenuation in total internal reflection takes place, for every predetermined time, thereby detecting whether the attenuation angle θsp changes, it is possible to know whether the analyte combines with the ligand or whether the analyte is a specific material to be combined with the ligand. As combinations of such an analyte and a ligand, there have been known combinations of antigens and an antibodies and of antibodies and other antibodies. For example, rabbit antihuman IgG antibody may be employed as the ligand with human IgG antibody employed as the analyte.
In order to detect the state of combination of the analyte in the buffer with the ligand, the total reflection attenuation angle θsp itself need not necessarily be detected. For example, a baseline is first measured by the use of a buffer containing no analyte, and then change of the attenuation angle θsp is measured when a buffer containing an analyte is added to the ligand, thereby measuring the state of combination of the analyte in the buffer with the ligand on the basis of the angle by which the attenuation angle θsp changes.
In the measuring system such as the surface plasmon resonance sensor, a method in which the measuring accuracy is improved on the basis of a reference method has been employed in order to cancel the measuring error due to external disturbance including a bulk effect due to the buffer, a temperature change of the ligand and/or the buffer or a change of the light source.
In the reference method, for instance, when the state of combination of the analyte with the ligand, two systems, one being a detecting system in which a ligand is fixed on the film layer and the other being a reference system in which no ligand is fixed on the film layer, are prepared. The result of detection of the detecting system is calibrated on the basis of the result of detection of the reference system, whereby the influence of the external disturbance is rejected. For instance, the result of detection of the reference system is subtracted from the result of detection of the detecting system.
However, it has been found that clear errors exist in the result of measurement even after calibration by the reference method. That is, there are some other factors of errors which cannot be calibrated by the reference method.